| dc.description.abstract | Bacteriophages are being reevaluated for their therapeutic potential, however, the pathways and mechanisms guiding genome transfer from the phage capsid to the host cytoplasm, a critical step in the phage infection cycle, remain unclear. Phages ensure their viral genome enters the host by traversing the host cell envelope. Phages infecting gram-negative bacteria, therefore, must contest the outer membrane, a periplasmic peptidoglycan meshwork, and the inner membrane to accomplish this task. While several canonical phages have evolved dedicated proteins and interaction pathways to facilitate penetration, less is known about how the smaller, simpler single-stranded (ss)RNA phages manage to penetrate the cell. The ssRNA phages universally infect their hosts by exploiting the mechanics of diverse, host-encoded retractile pili. These pili can facilitate significant biological functions, such as transfer of genetic material between cells, bacterial motility, and persistence. The purpose of the work outlined in this dissertation is to understand the function and role of pili and their respective machinery in guiding cell penetration among ssRNA phages. Genetic and fluorescence microscopy approaches yielded a foundational result: initial penetration of the viral payload causes a severance and release of surface pili from the host cell. This phenomenon was present in several paradigm phages, including the ssRNA coliphages MS2 and Qβ, interacting with conjugative F-pili, as well as PP7, a phage infecting Pseudomonas aeruginosa through type IV motility pili. For MS2, it was revealed that passage through the cell envelope requires the host-encoded coupling protein, TraD. For PP7, forces generated by an auxiliary retraction protein, PilU, was essential for cell penetration. Additionally, an experimental evolution approach was used to select for MS2 variants with improved plaquing to better understand how MS2 utilizes TraD for cell penetration. A single mutation in the maturation protein, responsible for pilus recognition, permitted plaquing in a cell background producing only basal levels of TraD. As well, the nucleotide binding activity of TraD was found to be essential for cell penetration by MS2. Lastly, quantitative analysis of F-pili secretion and dynamics was performed using several F-pilus machinery mutants to better understand how each gene affects the formation of conjugative pili. Many subunits of the pilus machine were found to be essential for pilus formation, while others promote pilus length and frequency. Overall, the work presented here provides a thorough examination of pilus-phage systems, exploring the pathways underlying pilus synthesis, function, and their role as facilitators of ssRNA phage cell penetration. | |
